Human cytomegalovirus (HCMV) is the major infectious cause of birth defects in the United States. Development of a vaccine to prevent HCMV-associated birth defects has been deemed a national priority. Two candidate vaccines are in Phase II trials - the Towne live attenuated vaccine and the gB/MF59 protein subunit vaccine. Both have been shown to elicit neutralizing antibodies that block HCMV entry into fibroblasts. However, recent work has revealed that HCMV enters endothelial, epithelial, and dendritic cells by an "endocytic" pathway that is quite distinct from the pathway used to infect fibroblasts. We found that the neutralizing activities of human immune sera are on average 48-fold more potent in neutralizing HCMV entry into epithelial cells compared to fibroblasts, suggesting that fibroblast-based assays reveal only a minor component of the HCMV-neutralizing response. Moreover, both Towne and gB/MF59 vaccines fell far short (28- and 15-fold, respectively) of natural infection in eliciting antibodies that neutralize HCMV entry into epithelial cells (Cui et al., Vaccine, 2008). Three HCMV proteins, UL128, UL130, and UL131 are essential for endocytic entry and compelling evidence suggests that all three proteins contain epitopes important for inducing antibodies that neutralize endocytic entry. These proteins are now under study as potential immunogens for induction of neutralizing antibodies that block viral entry into epithelial or endothelial cells. However, while the ability to elicit HCMV neutralizing antibodies can be measured in vitro, evaluating vaccine efficacy in preventing in utero transmission or fetal pathogenesis will require animal models. As cytomegaloviruses are strictly species-specific, animal models must utilize related cytomegaloviruses native to each species. Guinea pig cytomegalovirus (GPCMV) provides the only small animal model in which the ability of vaccines to protect against in utero transmission and/or fetal pathogenesis can be evaluated. The aims of this project are to determine: (1) if GPCMV can enter and replicate in epithelial or endothelial cells;and (2) if natural infection elicits neutralizing antibodies that specifically block endo/epithelial cell entry and if endo/epithelial entry occurs by a mechanism different from fibroblast entry. The answers to these questions will help determine if immunization strategies based on UL128, UL130, or UL131 can be evaluated in the guinea pig congenital infection model using the equivalent GPCMV proteins as immunogens. If so, the guinea pig model could provide important efficacy data that would help guide the design of new vaccines prior to human trials. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus is the major infectious cause of birth defects in the United States. We recently found that two current candidate vaccines fail to elicit high levels of antibodies that block viral entry into epithelial cells. Based on these results, new vaccines strategies are under investigation. This project aims to determine if guinea pig cytomegalovirus can be used to evaluate new vaccine strategies that are designed to elicit antibodies that block epithelial cell entry.